The present invention intends to provide a method to detect interactions between molecules, and particularly small molecules, which typically have a molecular weight of 1,000 Dalton or less, and biomolecular targets. The targets typically or generally are biological molecules involved in a disease state and the aim is to find interacting molecules which may turn out to be potential drug candidates.
In day-to-day laboratory practice, current methods of drug discovery rely primarily on “serendipity screening” wherein large libraries of low molecular weight chemicals are systemically added to a target molecule and assayed in vitro for binding or inhibition of, e.g., enzymatic activity, or in vivo for a biological response. If binding is the sought-after-function, then typically such screening programs seek compounds that bind with high affinity, for instance in the nanomolar to micromolar range, and at the same time high specificity. In order to increase the chances of finding such compounds, the sizes of the libraries used for screening have expanded to encompass a million or more compounds nowadays. Screening such libraries is clearly time-consuming and requires very large quantities of the target molecule.
More in detail, presently, the primary method of de novo drug discovery is based on adding essentially a random collection of chemicals to a pharmacologic target and assaying for binding. This method has been termed serendipitous screening. Since the likelihood of finding an interaction between two molecules is roughly inversely proportional to the desired affinity, libraries of up to a million compounds or more are now typically used to maximize the chance of finding interactions with the sub micro-molar affinity required of a drug. Tremendous technological advances in “high-throughout screening” (HTS) have allowed the process to be streamlined to the level that some systems can screen up to 100,000 compounds per day.
These systems are generally based upon measuring the signal generated when a fluorescent or radioactively labelled chemical probe is displaced from the target. Although highly efficient, these systems have a number of limitations. Almost all depend upon the a priori availability of a ligand in order to begin target screening. As a result, so-called orphan receptors, that is targets for which no ligand is available, are generally beyond the scope of HTS systems. In addition, because of the need to displace a pre-bound ligand, HTS is generally only sensitive to high affinity interactions. The high affinity lead compounds derived from HTS screening are typically complex and exhibit multiple interactions with the target. As a result, performing structure activity relationship (SAR) analysis on the large quantity of data from an HTS screen can be the limiting step. Furthermore, optimisation of these leads for enhanced specificity/affinity/oral availability can also be difficult.
In WO-A-97/18471 and WO-A-97/18469, a new approach to identify ligands to target biomolecules using NMR is described. The method described is termed SAR-by-NMR (“structure activity relationship”) and relies on the combination of serendipity screening and knowledge of the three dimensional structure of the target molecule. Instead of seeking both high affinity and high specificity ligands, the goal in these two applications is to find two non-mutually exclusive low affinity but high specificity ligands and chemically link these ligands together to form a high affinity ligand. NMR spectroscopy is used to detect the low affinity binding interactions, NMR being a technique ideally suited for that purpose. Changes in 2D-NMR spectra of the target molecule induced by ligand binding are detected.
More in detail, WO-A-97/18471 relates to a process for screening compounds to identify compounds that are ligands binding to a specific target molecule, said process is comprised of generating a 2D-15N/1H NMR correlation spectrum of a 15N-labelled target molecule; exposing the labelled target molecule to one or more chemical compounds; generating a second 2D-15N/1H NMR correlation spectrum; and comparing the two spectra to determine the differences.
WO-A-97/18469 discloses a process for designing a high affinity ligand, comprised of identifying at least two ligands to the target molecule, which bind to distinct binding sites on the target molecules, using multidimensional NMR spectroscopy; forming a ternary complex by exposing the at least two ligands to the target molecule; determining the 3D-structure of the complex formed and determining the spatial orientation of the at least two ligands; and using the spatial orientation determined to design the high affinity ligand.
The approach described in WO-A-97/18471 and WO-A-97/18469 was to use NMR to detect weak, but specific, interactions between the target, and more specific between a small, isotopically labelled protein, and the components of the compound library. The sensitivity of NMR to interactions of up to 1,000 fold lower affinity than required in the HTS methods means that in principle, 1,000 fold fewer compounds need be screened in order to equalize the chances of finding “hits”. In practise, the library for an NMR screen is typically 100 times smaller. In addition to its smaller size, a library used for the “SAR-by-NMR” approach consists of much simpler compounds than the libraries used for HTS, with molecular weights generally below 300 Da. In this light, reference is made to the two following scientific articles of the group of the inventors mentioned in both international applications: Hajduk et al. in Quarterly Reviews in Biosphysics 32 (1999) 211-240; and Hajduk et al. in Science 278 (1997), 12257-12261. The high specificity, low affinity lead compounds that result from such a screen can then be optimised by a variety of methods in order to produce realistic drug candidates.
SAR-by-NMR is a drug or ligand discovery system that can be carried out in its entirety by NMR. As such, it has a number of limitations. First, the target molecule must be available in large quantities (hundreds of mg to g) and must be isotopically labelled with either 15N or 13C, an impossibility with many targets. Second, the target should be less than roughly 30 kDa and be highly soluble and stable. Third, the resonance assignments for at least the backbone nuclei must be known and preferentially a 3D structure should be available. Furthermore, the target must be highly soluble and stable over a period of at least a few days to record the 2D-NMR spectra.
As an alternative approach to SAR-by-NMR, it is proposed to monitor the signals of the ligand instead of the target. Such an approach alleviates the constraints on molecular size and the necessity for isotopic labelling, but introduces other problems such as sensitivity and the need to re-optimize the system for every target. Additionally, large quantities of highly soluble target are still a prerequisite.
Such a method using NMR to identify ligands to target biomolecules is described in WO-A-98/57155. In this document, a method for identifying a drug core suitable for a given target is described, which method comprises providing a drug core consisting of a particular cyclic structure selected from a limited group. Thereto, the inventors of the invention described in this document propose to use one-dimensional solution NMR spectra to detect target-small compound interaction via changes in the spectrum of the small compound. The changes are detected via line broadening (a T2 relaxation phenomenon) diffusion filters or 2D transferred NOE (Nuclear Overhauser Enhancement) spectra.
Another such alternative method using NMR to identify ligands to target biomolecules is described in WO-A-98/48264. It uses simple and well-known 1H-NMR spectroscopy to detect low affinity binding events by observing changes in the NMR spectrum of the potential ligand. This method, like the method describe in WO-A-98/57155, does not provide the 3D structural information that the SAR-by-NMR method does, but it does avoid the limits of a requirement for isotopically labelled target. More in detail, this known method requires the steps of generating a first T2-relaxation filtered or diffusion-filtered proton-NMR spectrum of one or a mixture of chemical compounds; exposing the said one or more chemical compounds to the target molecule; generating a second T2-filtered or diffusion-filtered proton-NMR spectrum of the exposed one or more chemical compounds; and comparing the differences in the spectra recorded to identify the presence of one or more compounds which have bound to the target molecule.
The latter two prior art methods cannot overcome the need for large quantities of soluble target material. In particular, these methods exclude membrane bound proteins, which form a large and attractive class of pharmaceutically interesting target molecules.
Moreover, the method of WO-A-98/48264 can only be used when the target is sufficiently larger than the small compounds to be assayed. Although the inventors in said WO-A state that the target can be as small as 5 kDa, the present inventor has carried out experiments which indicate that the limit of sensitivity is closer to 10 kDa. In addition, it is noted that the sensitivity of the method described in WO-A-98/48264 varies with each target to be screened.
In general, it is noted that NMR has significant advantages as a detection method for serendipitous drug screening including high sensitivity to weak interactions and no requirement for a priori knowledge of a ligand. However, present NMR methods have several limitations. The most severe of these limitations is the requirement for large quantities of soluble and stable pharmacological targets.
It is a primary aim of the present invention to provide a method having the advantages of the known NMR screening techniques, but not having all or part of the disadvantages associated with the above-discussed techniques.